Degumming is an important step in oil refining, and the traditional hydration degumming suffers from high cost, large material consumption and serious environmental pollution, therefore, many have been committed to using enzymatic degumming in the degumming procedure for oil refining and great progress has been made in recent years. Compared with traditional methods, enzymatic degumming can improve economic efficiency, save energy, reduce emission, decrease environment pollution, and have larger advantages in terms of environment, economic and quality. The enzyme used in oil degumming is phospholipase. Phospholipases possess the ability to hydrolyze one or more ester bonds of phosphoglyceride and represent a class of lipases, acyl hydrolases and phosphatases. Phospholipases, depending on its site of action in the phospholipid molecule, can be divided into phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC) and phospholipase D (PLD).
Phospholipase C (for short, PLC), is a lipid hydrolase capable of hydrolyzing C3 phosphatidyl site of glycerophospholipids to form diacylglycerol and phosphorylcholine, inositol phosphates, phosphoethanolamine and other. Phospholipase C is widely found in plants and microorganisms. Plant and animal derived PLCs generally locate on cell membrane, which are complicated in structure, belonging to endogenous phospholipase C, and difficult to separate. Compared to other degumming enzymes, phospholipase C (PLC) showed greater advantages, such as increased yield of diacylglycerol (DAG), and reduced lose of obtained oil.
Microbial derived PLCs generally have simpler structures, and these enzymes have been isolated from various microorganisms, including many bacterial origin comprising Clostridium perfringens [Yun T, Siebel C. Cloning and expression of the PLC gene from Clostridium perfringens and Clostridium bifermentants[J]. Infection and immunity, 1989, 2: 468-476], C. bifermentans, Burkholderia pseudomallei, Bacillus cereus, Bacillus mycoiddes, Bacillus thuringiensis, Listeria monocytogenes, Pseudomonas aeruginosa, P. fluorescens, Straphylococus aureus, Acinetobacter baumannii, Streptomyces clavuligerus, Burkholderi, and others. They may be from actinomycetes such as Streptomyces hachijyoensis, and others. They may also be from yeasts such as Candia albicans [Analuz E, Juan-Jose R, Rosario Cueva. Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinostiol-phospholipase C[J]. Yeast, 2001, 18(8): 711-721], Saccharomyces cerevisiae [Payne W, Fitzgerald-Hayes M. A mutation in PLC1, a candidate phosphoinositide specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation[J]. Molecular and Cellular Biology, 1993, 13: 4351-4364], and others.
PC-PLC from Bacillus cereus (BC-PC-PLC) is an earlier studied phospholipase C. BC-PC-PLC has a full length of 283 amino acids, comprising a signal peptide of 24 amino acids and a leader peptide of 14 amino acids. The mature form thereof has 245 amino acids (Johansen, T., Holm, T., Guddal, P. H., Sletten, K., Haugli, F. B., Little, C, 1988, “Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus”, Gene 65(2): 293-304). The crystal structure of BC-PC-PLC has been reported, which consists of a plurality of helical domains and has a catalytic site of aspartic acid 55 and at least three Zn2+ binding sites (Hough., E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E., Derewenda, Z., 1989, “High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus”, Nature, 338:357-60). Little has been studied about the heterologous expression of BC-PC-PLC other than that in Bacillus subtilis and pichia pastoris (Durban, M. A., Silbersack, J., Schweder, T., Schauer, F., Bornscheuer, U. T., 2007, High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis, Appl Microbiol Biotechnol 74(3):634-639; Seo, K. H, Rhee J. I., 2004, High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization, Biotechnol Lett 26(19):1475-1479).
Currently, phospholipase C is mainly used in enzymatic degumming. In the manufacture of edible oil such as soybean oil and rapeseed oil, unrefined crude oil mainly comprises a complex mixture of triglycerides, phospholipids, sterols, tocopherols, free fatty acids, trace metals and other trace compounds, wherein the phospholipid will cause deterioration of color and taste, shorter shelf life and affect the effect of subsequent refining process. Currently, the main degumming processes comprise hydration degumming, deep degumming and enzymatic degumming. Enzymatic degumming is becoming preferred due to its mild condition, non-pollution, and low oil consumption.
Since phospholipase C can act on glycerophospholipids to generate diacylglycerol, use of phospholipase C in the enzymatic degumming process thus can significantly improve the yield of oil, thereby enhancing the economic efficiency of production. Therefore, it is of important practical significance to improve the degumming performance of phospholipase C.
However, there is still need of BC-PC-PLC with a higher enzymatic activity in the field.